﻿Unveiling the identity of Diaurora Cockerell, 1903 (Bivalvia, Unionidae): morphology, molecular phylogenetics, and the description of a new species

﻿Abstract The monotypic freshwater mussel genus Diaurora Cockerell, 1903 has long been enigmatic due to its rarity and morphological confusion with Acuticosta. In this study, we comprehensively redescribed Diauroraaurorea (Heude, 1883) through a detailed analysis of shell morphology and molecular phylogenetics of recently collected specimens. Moreover, a new species, Diauroralaevesp. nov., was identified from the Fuyishui River, a tributary of the Zishui River in Shaoyang County, Shaoyang City, Hunan Province, China. Molecular phylogenetic analyses showed that D.aurorea and D.laevesp. nov. were reciprocally monophyletic and formed a clade as sister to Schistodesmus. Our study underscores the necessity of further exploring the diversity of freshwater mussels in understudied small tributaries throughout China.


Introduction
Freshwater mussels, an important component of freshwater ecosystems (Liu et al. 1979;Vaughn and Hoellein 2018;Lopes-Lima et al. 2021), represent a diverse group of bivalve mollusks that are often identified by their unique shell morphology (Lopes-Lima et al. 2017;Chernyshev et al. 2020). However, reliance on morphological characteristics for taxonomy has resulted in some confusion within the group, as some species may share similar morphological characteristics and exhibit phenotypic plasticity (Huang et al. 2018b;Wu et al. 2020;Wu et al. 2022). One such example is Diaurora Cockerell, 1903, a monotypic genus of small-sized freshwater mussels that are endemic to the middle and lower reaches of the Changjiang River, also known as the Yangtze River (Heude 1883;Simpson 1900;Wu et al. 2000;Shu et al. 2009;He and Zhuang 2013;Guo 2022).
Initially, Simpson (1900) described the genus as a subgenus of Parreysia Conrad, 1853, based on the original description of Unio auroreus Heude, 1883, and later renamed as Diaurora by Cockerell (1903). Unio auroreus was originally collected and described by Heude (1883) based on the holotype USNM 472411 (designated by monotype) from Ning-kouo hien in China. However, only a few additional specimens have been reported sporadically since its original description, rendering this species little-known and enigmatic for over a century. Due to the limited availability of specimens for study and the potential confusion arising from its easily confused morphology with Acuticosta Simpson, 1900, the validity and systematic status of Diaurora have remained controversial for an extended period. It has been treated as a separate genus (Haas 1924), a subgenus of Parreysia Conrad, 1853(Simpson 1900, or even a synonym of Acuticosta (Wu et al. 1999).
To address these taxonomic uncertainties, we analyzed the validity and systematic status of Diaurora using a combination of shell morphology and molecular phylogeny. Furthermore, we redescribed the enigmatic species Diaurora aurorea and described a new species, Diaurora laeve sp. nov., from the Zishui River of Hunan Province, China. Our study sheds light on the distribution, ecology, and protection of this distinct genus, and highlights the possibility of small Chinese tributaries harboring unique freshwater mussels.

Specimen collection and identification
Specimens were collected by hand net from the tributaries of the Changjiang River and the Jiulongjiang River from 2003-2022. These specimens were fixed in 95% ethanol, and some specimens were dissected for observation of the internal structure. Measurements were taken point-to-point with digital calipers recorded to the nearest 0.1 mm.

Molecular phylogenetic analyses
Genomic DNA was extracted from somatic tissues preserved in 95% ethanol using a TIANamp Marine Animals DNA Kit (Tiangen Biotech, China). The quality and concentration of the DNA were checked on 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, USA). F-type mitochondrial COI sequences, which are considered the barcode marker, were amplified using primers LCO22me2 (GGTCAACAAAYCATAARGATATTGG) and HCO700dy2 (TCAGGGTGACCAAAAAAYCA) (Walker et al. 2007). Polymerase chain reaction (PCR) amplifications of COI were performed in a final 25 μL volume mixture containing 1 μL of template DNA, 1 μL of each pair of primers, 12.5 μL of Green Taq Mix (Vazyme, China), and 9.5 μL ddH 2 O. Thermal cycling began with one cycle at 98 °C for 10 s, followed by 35 cycles of denaturation at 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min, with a final extension step at 72 °C for 7 min. The PCR products were purified and sequenced using an ABI 3730XL analyzer by Sangon Biotech (China). The accession numbers of all newly obtained sequences are given in Table 1.
Sequences were aligned using MEGA v. 6.0 (Tamura et al. 2013) and checked by eye. The genetic distance, based on the uncorrected p-distance model, was calculated using MEGA v. 6.0. Phylogenetic relationships were reconstructed using maximum likelihood (ML) and Bayesian inference (BI). Thirty-six reliable unionid sequences from GenBank were use for phylogenetic analysis. Margaritifera dahurica (Middendorff, 1850) and Gibbosula rochechouartii (Heude, 1875) were used as the outgroup for rooting the tree. ML analyses were performed in IQ-TREE v. 1.6.12 (Minh et al. 2013) with 1000 reiterations. The most appropriate model of sequence evolution (TIM3+I+G) was selected under PartitonFinder2 v. 1.1 (Robert et al. 2017). Bayesian inference (BI) was conducted in MrBayes v. 3.2.6 (Ronquist et al. 2012). The most appropriate model of sequence evolution (GTR+I+G) was selected under ModelFinder (Subha et al. 2017). Four simultaneous runs with four independent Markov Chain Monte Carlo (MCMC) were implemented for 10 million generations, and trees were sampled every 1000 generations with a burn-in of 25%. The convergence was checked with the average standard deviation of split frequencies <0.01 and the potential scale reduction factor (PSRF) ~1.

Phylogenetic relationships
A total of 43 mitochondrial COI sequences from 38 unionoid species were utilized in the phylogenetic analyses (Table. 1). Phylogenetic analyses revealed ML and BI trees with largely consistent topologies (Figs 1, 2). Our results showed that Diaurora occupied a distinct position in the subfamily Unioninae and was the sister group of Schistodesmus Simpson, 1900 instead of Acuticosta. However, this sister relationship between Diaurora and Schistodesmus was not well-supported (bootstrap support value = 70 and Bayesian inference posterior probability support value = 0.65). Furthermore, Diaurora can be divided into two separate clades: one consisted of Diaurora aurorea, and the other represents a yet-to-bedescribed species with distinct morphological characters. The genetic distance (uncorrected p-distance) between the two clades in Diaurora was 10.4%. Indeed, we acknowledged that deep relationships in our phylogenetic trees based on the COI dataset were not well resolved but should not overturn the main conclusion drawn here. It is necessary to further reveal the exact phylogenetic relationships using more data such as mitochondrial genomes in the future.
Remarks. Aurora Simpson, 1900 was originally described as a subgenus of Parreysia based on shell morphology. Simpson (1900) indicated uncertainty regarding the taxonomic placement of Unio auroreus and whether it merited a generic rank. As a result, he provisionally designated it as a new subgenus. Cockerell (1903) renamed Aurora as Diaurora due to the former being a junior homonym of Aurora Ragonot, 1887 (Insecta: Lepidoptera: Pyralidae) and Aurora Sollas, 1888 (Porifera: Demospongiae: Ancorinidae). Haas (1924) elevated Diaurora to an independent genus, but this viewpoint was not widely accepted. Wu et al. (1999) recombined Parreysia aurorea as Acuticosta aurorea based on shell morphology and marsupium type. While Diaurora shares some similarities in shell morphology with Acuticosta, it can be differentiated from Acuticosta by its distinct shell shape and the absence of a posterior ridge (Fig. 3). ( Diagnosis. Shell triangular-ovate; posterior margin obliquely arc-shaped. Periostracum with straight broken blackish-green rays. Zigzag sculpture presented on all over the shell surface.

Diaurora aurorea
Description. Shell (Figs 3A, 4A). Shell small size, symmetric, solid, moderately thick, sub-glossy, triangular-ovate. Anterior margin oval, inflated; dorsal margin curved downwards; ventral margin slightly rounded or nearly straight; posterior margin obliquely arc-shaped. Umbo inflated, above hinge line, located at 1/3 of the dorsal margin, and often eroded. Periostracum orangish to brownish, with straight broken blackish-green rays and thin growth lines. Growth lines arranged in irregular concentric circles. Zigzag sculpture presented on all over the shell surface but weakening from umbo to edge. Hinge short. Ligament short and strong. Mantle muscle scars obvious. Anterior adductor muscle scars oval, deep, smooth in junior but rough in adult; posterior adductor muscle scars long oval, smooth. Left valve with two pseudocardinal teeth, equal height, anterior tooth small and flat, posterior tooth thick and pyramidal; anterior pseudocardinal tooth of the right valve well developed, posterior pseudocardinal tooth reduced, connected to lateral teeth. Lateral teeth of both valves long and thick. Nacre white or light orangish.
Remarks. The rarity of D. aurorea has been seriously underestimated for a long time due to the common misidentification of museum material. Upon examination of museum specimens, it has been discovered that specimen NZMC10542 in the National Zoological Museum of China and specimens 15_NCU_XPWU_AA01-11 in Nanchang University were actually Acuticosta chinensis. Since its original description, only a handful of additional specimens of D. aurorea have been correctly identified. It has always been shrouded in mystery as there are hardly any photographs of specimens other than the type specimen. The new specimens examined in this study indicate that it is far more widespread than previously recorded. However, the habitats of D. aurorea are severely fragmented and the population size is very small. Therefore, it requires more attention and protection in the future. Diagnosis. Shell reniform. Periostracum with irregular broken blackish-green rays. Zigzag sculpture only presented in umbo area.

Diaurora laeve
Description. Shell (Figs 3B, 4B). Shell small size, symmetric, solid, moderately thick, sub-glossy, reniform. Anterior margin oval, inflated; dorsal margin curved downwards and truncate; ventral margin slightly rounded or nearly straight; posterior margin oval. Umbo inflated, above hinge line, located at 1/3 of the dorsal margin, and often eroded. Periostracum orangish to brownish, with irregular broken blackish-green rays and thin growth lines. Growth lines arranged in irregular concentric circles. Zigzag sculpture only presented in umbo area. Hinge short. Ligament short and strong. Mantle muscle scars obvious. Anterior adductor muscle scars oval, deep, smooth in junior but rough in adult; posterior adductor muscle scars long oval, smooth. Left valve with two pseudocardinal teeth, equal height, anterior tooth small and flat, posterior tooth thick and pyramidal; anterior pseudocardinal tooth of the right valve well developed, posterior pseudocardinal tooth reduced, connected to lateral teeth. Lateral teeth of both valves long and thick. Nacre light orangish.
Soft anatomy (Fig. 5B). Mantle off-white to light-brownish, aperture margins brown, flap margin with yellowish papillae. Gills light-brownish, inner gills slightly longer and wider than outer gills. Labial palps yellowish to brown, distally pointed and irregularly fan-shaped in appearance. Visceral mass creamy white, foot orange.
Etymology. The specific name laeve is made from the Latin laeve for smooth, an adjective, alluding to the smoother shell surface of this species. Vernacular name. 平滑金黄蚌 (Pinyin: ping hua jin huang bang). Distribution and ecology. Diaurora laeve sp. nov. is known from the type locality only (Fig. 6). It was found to occur in a pebbly substrate of the river together with Nodularia douglasiae and Lanceolaria triformis (Fig. 7). It is the dominant species in the habitat, accounting for 98% of the total density of freshwater mussels.  22_NCU_XPWU_DL50. aam, anterior adductor muscle; pam, posterior adductor muscle; ea, excurrent aperture; ia, incurrent aperture; pia, papillae of the incurrent aperture; pea, papillae of the excurrent aperture; ig, inner gills; og, outer gills; m, mantle; lp, labial palps; vm, visceral mass; f, foot.  Remarks. The placement of the new species in Diaurora is supported by both morphology and molecular phylogenetic analysis. Diaurora laeve sp. nov. can be easily distinguished from D. aurorea by its reniform shell, smaller zigzag sculptured area, and irregular rays on the shell. The different habitat preferences of Diaurora laeve sp. nov. and D. aurorea may have led to their differentiation. Diaurora aurorea is commonly found in the middle reaches of sandy substrate tributaries, while Diaurora laeve sp. nov. prefers to inhabit the upper reaches of pebbly substrate tributaries. In recent years, with more in-depth investigations and field surveys, new freshwater mussel species such as Inversidens rentianensis Wu & Wu, 2021 and Pseudocuneopsis sichuanensis Huang, Dai, Chen & Wu, 2022 have been discovered in small tributaries of China (Wu et al. 2021;Wu et al. 2022). These findings suggest that the diversity of freshwater mussels in small tributaries of China remains to be fully explored, and future extensive exploration may lead to the discovery of other yet-to-be-described species.